Glatiramoids are a family of synthetic copolymers containing four amino acids: L-glutamic acid, L-alanine, L-lysine and L-tyrosine, in a defined molar ratio (Varkony et al. 2009). Glatiramer acetate (GA) is a member of the glatiramoid family, and it is the active pharmaceutical ingredient of the commercially available medicine Copaxone® (Teva Pharmaceutical Industries Ltd., Israel), indicated for the treatment of patients with relapsing forms of multiple sclerosis.
According to the product labeling, GA consists of the acetate salts of synthetic polypeptides, containing four naturally occurring amino acids: L-glutamic acid, L-alanine, L-tyrosine, and L-lysine with an average molar fraction of 0.141, 0.427, 0.095, and 0.338, respectively. The average molecular weight of GA ranges from 5,000 to 9,000 daltons.
Chemically, GA is designated L-glutamic acid polymer with L-alanine, L-lysine and L-tyrosine, acetate (salt). Its structural formula is:(Glu,Ala,Lys,Tyr)x.xCH3COOH(C5H9NO4.C3H7NO2.C6H14N2O2.C9H11NO3)x.xC2H4O2                 CAS—147245-92-9        (NDA 020622/S-089 FDA Approved Labeling Text dated Jan. 28, 2014)        
GA is synthesised via amino acid polymerisation followed by a subsequent cleavage or partial depolymerisation step. Because of the stochastic nature of polymerisation and cleavage reactions, the polypeptides obtained by this process vary in sequence, length and molecular weight (MW), resulting in a characteristic molecular weight distribution (MWD) that spans from about 2,500 to 20,000 daltons.
The average MW and the MWD represent important characteristics to distinguish GA from other glatiramoids or similar polypeptide mixtures. In fact, differences in the MWD profile may be both indicative of a different manufacturing process and partially predictive of a different biological activity. When compared to GA, high MW copolymers are highly immunogenic and toxic, whereas low MW glatiramoids show lower biological potency in pharmacological models (Ramot et al. 2012).
The MWD, or molar mass distribution, of a polymer describes the relationship between the number of moles of each polymer species (Ni) and the molar mass (Mi) of that species. The MWD may be represented either graphically, i.e. as a cumulative or differential curve (see FIGS. 5 and 6), or in a numeric way, as a table where the cumulative MWD is represented reporting the mass percentage of the macromolecules having molecular weight smaller than or equal to a specified value (see Table 1).
Alternatively, the MWD may be represented by calculating the most common molecular weight averages. Different average values, or moments, can be defined, depending on the statistical formula that is applied. In practice, at least three averages are used:                Number average molar mass: Mn=Σ(NiMi)/ΣMi         Weight average molar mass: Mw=Σ(NiMi2)/Σ(NiMi)        Z-average molar mass: Mz=Σ(NiMi3)/Σ(NiMi2)        Dispersity: D=Mw/Mn         
Where Ni is the number of molecules with molar mass Mi.
If the polymer is a homogeneous (“monodisperse”) sample, then all the molecular weight averages are equivalent. In the case of a heterogeneous (“polydisperse”) polymer sample, the ratio of Mw to Mn (D=Mw/Mn) and that of Mz to Mw (Mz/Mw) are measures of the dispersity (heterogeneity) of the MWD.
Size exclusion chromatography (SEC) is a chromatographic technique that separates molecules in solution by their molecular size, or more precisely, by their hydrodynamic volume. The SEC stationary phase is a porous material with a characteristic pore size distribution, and depending on molecular size, a larger or lesser fraction of the pores is accessible to the macromolecules. Accordingly, the elution volume of a solute increases as its molecular size decreases. When properly calibrated, a SEC system is the most convenient method for the determination of the overall MWD of a polymer such as GA.
Two different strategies or methods are described in patent literature for obtaining the molecular weight distribution of a polypeptide mixture via SEC. The first group of methods provides values of molecular weight based on a calibration obtained with a set of monodisperse standards. These standards are either single molecules (obtained by synthesis or recombinant DNA technology) or mixtures with narrow MWD obtained by fractionation of a glatiramoid sample and in all cases are different from real GA.
A second group of methods provides “absolute” values of the molecular weight generally using a molecular weight sensitive detector, e.g. a Right-Angle Light Scattering (RALS), a Low-Angle Light Scattering (LALS), RALS/LALS hybrids or a Multi-Angle Light Scattering (MALS), usually combined with a concentration detector, either based on refractive index (RI) or ultraviolet absorption (UV).
There are numerous patents (U.S. Pat. No. 6,514,938, U.S. Pat. No. 6,800,287, U.S. Pat. No. 7,074,580, U.S. Pat. No. 7,163,802, U.S. Pat. No. 7,615,359 and U.S. Pat. No. 8,399,211) teaching how to use standards made of single polypeptide molecules for the calibration of SEC columns employed in the determination of MWD of batches of GA. The need of using a set of dedicated standards depends on the particular structural characteristics of GA polypeptides. SEC fractionation is regulated by molecular hydrodynamic volume, so the MWD of a copolymer can be accurately estimated only if the MW standards have the same higher order structure—and thus, the same density or partial specific volume—of the sample (S. Mori and H. G. Barth Size Exclusion Chromatography, Springer 1999, pp. 101-104). For example, using a commercial mixture of proteins as the MW standard, the obtained apparent MW of GA resulted to be higher than that one obtained using a set of GA-like peptides (see patent application US 2007/059798). This is because globular proteins are more structured than GA polypeptides, thus having a higher density (or lower partial specific volume). Accordingly, the use of “normal” commercially available protein markers is not appropriate for the determination of the MWD of GA batches.
Nonetheless, any set of polypeptide standards, either consisting of single molecules or mixtures with narrow MWD, will still be different from the GA mixture, that contains a huge number of polypeptides of different sizes that completely covers the entire range of molecular weights of the MWD. Thus, the MWD assessed by these methods can only be relative, not absolute. Moreover, it is evident that the preparation of sufficient quantities of long, pure peptides (either by chemical synthesis or by recombinant DNA technology) can be very laborious and costly.
The other approach, based on absolute SEC methods, has been recently suggested in the patent application US 2014/0045740, herein incorporated by reference: according to that teaching, every GA batch can be analysed using SEC chromatography coupled to multi-angle light scattering and refractive index detectors (SEC-MALS-RI). Although the method is “absolute” and overcomes the need for column calibration, this technique is unsuitable to be applied routinely for quality control and/or batch release purposes. Indeed, the MALS detector is subject to some practical problems: (a) a low signal to noise ratio, due to the presence of extraneous particles; (b) the high sensitivity of the MALS signal to even small and reversible aggregation phenomena. These practical problems reduce the repeatability and reproducibility of the MWD analysis by means of the SEC-MALS-RI method.
It is thus evident the need for a more practical and robust analytical method useful for determining the detailed and accurate MWD of GA and other GA-like polypeptide mixtures. Accurate evaluation of the MWD via such analytical methods is crucial for the choice of GA batches suitable for pharmaceutical use.